There is an urgent need for early-detection cancer biomarkers which can significantly lower the high mortality and cost of treating cancer. One of the most promising candidates for such biomarkers is tumor-associated autoantigens (TAA), autoantibodies to which have been detected in patient's blood even in the early stages of cancer. In addition to use in early diagnosis, such TAAs have great value in developing anti-cancer vaccines and potentially guiding individualized patient therapy. One general approach to TAA discovery is screening the sera of patients having particular cancers against proteome-scale libraries of human proteins, to detect autoantibody binding. However, large-scale proteomic screens are difficult to perform using conventional technology, such as protein microarrays, due to limited sensitivity, reproducibility, scalability and expense of manufacture. An additional problem is the reliance on attachment and drying of the bait protein onto planar microarray substrates, which damages proteins, impairs the subsequent bio-assay kinetics and reduces the amount of accessible bait protein. During Phase I, we have made significant progress towards overcoming these limitations through the development of a next generation approach to proteomics, termed Bead-based Global Proteomic Screening (Bead-GPSTM). Bead-Protein libraries are produced inexpensively and rapidly using cell-free protein expression and uniformly attached to beads. Bio-assays are performed with the beads in suspension for improved kinetics. The bead library is then randomly arrayed into Pico-well slides with a capacity of up to 1-million beads. The strength of positive bait-prey interactions on the bead is quantitatively measured using fluorescence imaging and then the bead identity decoded using photocleavable mass tags combined with MALDI mass spectrometry imaging (MALDI-MSI), which provides the ability to decode tens of thousands of different bead species. During Phase I we have achieved all key milestones including: i) full production and validation of a prototype 100- member protein library based on cell-free expression methods, and, formatting the library on beads; ii) development and evaluation of an ultra-high density bead-array platform including decoding using MALDI-MSI; iii) successful screening of the prototype bead-protein library with several model autoantigens; iv) discovery and validation of a new TAA for CRC on the Bead-GPSTM platform by using candidate pre-selection with ultra-low noise gene expression profiling; v) demonstration of a MALDI-MSI bead barcoding system capable of generating tens of thousands of unique codes using <50 distinguishable mass tags; vi) successful screening of 94 CRC sera and controls using a model bead-protein library on Illumina's mid-plex automated VeraCodeTM platform. During the Phase II project we will continue to develop and evaluate Bead-GPSTM with special focus on the discovery of TAA biomarkers for colorectal cancer (CRC). For this purpose we will initially fabricate an intermediate 500-member library including known CRC TAAs from the literature, high probability TAAs and those discovered in Phase I. The library will be screened using Bead-GPSTM against CRC patient sera and matched controls including individuals with benign polyps and benign inflammatory diseases. Rigorous SOPs for serum collection/handling developed by an NCI (EDRN) consensus working group for biomarker discovery will be implemented by both our commercial (ABS) and institutional (Boston Medical Center) suppliers (see letters), and assisted by consultant M. Tuck, who helped spearhead the EDRN effort (see letter). For comparison, similar measurements will be run using the VeraCodeTM digital holographic bead technology in collaboration with Illumina Inc., who has agreed to provide the instrumentation (BeadXpress) and reagents (see letter; note that AmberGen has won the Illumina Assay Design Challenge for Proteomics on VeraCode beads). The 500-member protein library will be followed by a 5,000-member library later in Phase II. To accelerate commercialization, we will work closely with Illumina in order to implement clinical multipanel assays on its VeraCodeTM system, as well as with INOVA and Bio-Rad Diagnostics, two leaders in development of clinical immunoassays (see letters). Finally, we will work closely during Phase II with Bruker Daltonics Inc., which has expressed interest in developing an integrated instrument for Bead-GPSTM.